Cell surface receptors are an important class of proteins involved in cellular functioning because they are the primary mediators of cell to cell communication. For example, G protein coupled receptors (GPCRs) are an important category of cell surface receptors. The medical importance of these receptors is evidenced by the fact that more than 60% of all commercially available prescription drugs work by interacting with known GPCRs.
In their resting state, the G proteins, which consist of alpha (α), beta (β) and gamma (γ) subunits, are complexed with the nucleotide guanosine diphosphate (GDP) and are in contact with the receptors to which they are coupled. When a hormone or other first messenger binds to receptor, the receptor changes conformation and this alters its interaction with the G protein. This causes the α subunit to release GDP, and the more abundant nucleotide guanosine triphosphate (GTP) displaces it, activating the G protein. The G protein then dissociates to separate the α subunit from the still complexed beta and gamma subunits. Either the Gα subunit, or the Gβγ complex, depending on the pathway, interacts with an effector. The effector (which is often an enzyme) in turn converts an inactive precursor molecule into an active “second messenger,” which may diffuse through the cytoplasm or may be associated with downstream signal molecules, triggering a signal cascade. After a few seconds, the Gα converts the GTP to GDP, thereby becoming inactive. The inactivated Gα may then reassociate with the Gβγ complex.
Hundreds, if not thousands, of receptors convey messages through heterotrimeric G proteins, of which at least 17 distinct forms have been isolated. Most G protein-coupled receptors are comprised of a single protein chain that is threaded through the plasma membrane seven times. Such receptors are often referred to as seven-transmembrane domain receptors (STRs). More than a hundred different GPCRs have been found, including many distinct receptors that bind the same ligand, and there are likely many more GPCRs awaiting discovery.
The mating factor receptors of yeast cells (STE2 and STE3) also span the membrane of the yeast cell seven times and are coupled to yeast G proteins. Heterologous GPCRs can be expressed in yeast cells and can be made to couple to yeast G proteins resulting in the transduction of signals via the endogenous yeast pheromone system signaling pathway which is normally activated by STE2 or STE3. In some cases, such heterologous receptors can be made to couple more effectively to the yeast pheromone system signaling pathway by coexpressing a heterologous G protein α subunit (e.g. U.S. Pat. No. 5,482,835 of King et al), by expressing a chimeric G protein subunit (e.g. WO 94/23025), or by expressing a chimeric G protein coupled receptor (e.g., U.S. Pat. No. 5,576,210 of Sledziewski et al.).
The βγ subunits of the activated G protein stimulate the downstream elements of the pheromone system pathway, including the Ste20p protein kinase, and a set of kinases that are similar to MEK kinase, MEK (MAP kinase kinase), and MAP kinase of mammalian cells and are encoded by the STE 11, STE7, and FUS3 genes, respectively (Whiteway et al. 1995. Science. 269:1572).
In recent years drug discovery has been advanced by expression of heterologous receptors in living cells. However, due to the complexities inherent in such heterologous expression studies, the development of reliable assays to search for modulators of these receptors has presented particular challenges. For example, it is often difficult to obtain sufficient signal amplification to enable efficient identification of modulatory agents. The development of new drug discovery assays to identify novel modulators of receptors (e.g., GPCRs) would be of tremendous benefit.